Endotoxin, a lipopolysaccharide (hereinafter “LPS”), is originated from cell wall of most gram-negative bacteria including Escherichia coli, and often present in a large quantity during protein production. Since the presence of a small amount of endotoxin can cause severe inflammation and septic shock on animals, the LPS level of a protein drug shall be lower than 10 EU/mL to ensure safe usage of the drug via intravenous injection.
Current approaches to endotoxin reduction are mainly based on electrostatic interaction between positively charged adsorbents and negatively charged endotoxins. For examples, filtration, ion exchange chromatography, Triton X-114 phase separation, cationic polymer adsorbents like histamine- or histidine-immobilized Sepharose and dextran-coated particles are common methodologies. However, these methods have poor selectivity in removing endotoxins from protein solutions containing other acidic proteins such as bovine serum albumin (BSA). The poor selectivity is due to the fact that acidic proteins and endotoxins have the same negative charges at neutral pH, thus adsorption through electrostatic interaction cannot distinguish different acidic proteins, resulting in a significant loss of the desirable acidic proteins.
In order to selectively remove only the LPS, but not other proteins in a protein mixture, Miltenyi Biotech. has developed a type of magnetic particles that are coated with polycationic ligands (detailed chemical compositions are not disclosed). It is claimed that the particles could selectively adsorb LPS from BSA solution with the LPS removal efficiency up to 99% and the BSA recovery of 96%. However, such high selectivity can only be achieved at a relatively low buffer strength (e.g. 0.1 M phosphate buffer at pH 7) with a specific buffer type (phosphate buffer). For other buffer solutions such as in 50 mM Tris buffer at pH 7, although high LPS removal efficiency could still be attained, the protein recovery is substantially reduced to 67%. Another approach to achieve selective removal of LPS is the use of polymyxin B-immobilized column. Its selective property is attributed to the hydrophobic interaction between the lipid A and LPS molecules. However, this method suffers from a significant protein loss when a small sample volume is used. A microfiltration membrane made of a Nylon immobilized with cationic ligands such as polymyxin B, poly-L-lysine and poly(ethylenimine) on its surface have also been used to selectively remove LPS. Although the polymer-coated membranes can give a satisfactory adsorption capacity and show selectivity for endotoxins, their good performance is restricted to low salt concentrations (e.g. 0.02 M phosphate buffer). In addition, the leakage problem of polymyxin B during LPS adsorption process is of a serious concern because the polymyxin B is neuro- and nephrotoxic to mammals. Another concern for this method is the filter blockage which is a common problem associated with the use of microfiltration membrane. Therefore, methodology which can selectively and effectively remove endotoxins in the presence of both acidic or basic protein solution under a broad range of pH, salt concentration and type of buffer solution (e.g. 1×PBS, blood containing buffer) is still of a great demand.
Chisso Corp. disclosed an adsorbent which is made of polylysine-immobilized cellulose porous microspheres (Chisso Corp.: Cellufine™ ET clean). These adsorbents show satisfactory LPS adsorption capacity and selectivity under physiological buffers (ionic strength from 0.1 to 0.5 M, pH 7). The success of these materials contribute to two key features: the presence of selective-endotoxin binding ligand polylysine and the porous structure. The presence of porous structure provides a large surface area and appropriate pore size to selectivity adsorb and trap the LPS molecules since the endotoxins molecules are small enough to diffuse into the particle pores, and retain inside the microspheres. The larger size proteins are quickly eluted due to their sizes larger than the exclusion limit of the microspheres pores. However, since the porous cellulose microspheres are prepared by a random suspension cross-linking method, their particle and pore sizes are not uniform, resulting in poor reproducibility. In addition, this method involves a multi-step surface modifications and the use of expensive reagents such as polylysine. Another problem often encountered with the use of porous materials is the slow LPS adsorption kinetics because LPS has to diffuse from exterior environment into the pores of the microspheres for adsorption. Thus, the method is quite time-consuming (2 hour incubation time is required for a batch method). When the microspheres are packed into a column as an affinity chromatography, slow flow rate must be maintained (between 0.17 and 0.5 mL/min) in order to achieve high removal efficiency of the LPS.
Recently, Yuan et al. reported that adsorption capacity of endotoxins could be increased almost 8 times at pH<7 when using an adsorbent containing a ligand with hydroxyl group at b position adjacent to quaternary ammonium ion. The significant improvement is attributed to the strong hydrogen bonding between the hydrogen atom of phosphate group of the endotoxin molecule and the oxygen atom of the β-hydroxyl group. The hydrogen bonding results in the formation of an octatomic ring. However, this method only gives barely satisfactory LPS adsorption efficiencies due to the low grafting density of the ligand on the adsorbent. In addition, the optimal working pH for the LPS adsorption must be below pH 7, thus limiting the scope of proteins.
Therefore, there remains a strong need for a new method of endotoxin removal which overcomes the problems as described above.